FLUORESCENCE STUDY OF TRP IN CHEY AFTER T-JUMP

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我们提出利用色氨酸荧光和T-jump技术研究CheY与CheY结合域FliM对应的16个残基肽的复合物。当CheY未磷酸化时，肽残基与CheY的亲和力较弱，而在磷酸化状态下，其亲和力约为25倍。发现单个色氨酸的固有色氨酸荧光在肽结合时被猝灭，这种信号丢失可以在动力学实验中用于监测结合。以前使用核磁共振，一个建议的机制可能涉及到一个主要的非活性构象和一个能够结合肽的少数活性构象之间的异构化。在这种观点下，磷酸化的作用是改变活性和非活性形式之间的平衡。我们建议使用激光t跳技术来监测静息和磷酸化状态下的结合动力学。我们还建议研究一组具有活性和非活性构象之间改变平衡的突变体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We propose to study the complex of CheY with a 16 residue peptide corresponding to the CheY binding domain FliM using tryptophan fluorescence and T-jump technology. The affinity between the peptide residue and CheY is weak when CheY is unphosphorylated and about 25 times stronger in the phosphorylated state. The intrinsic tryptophan fluorescence of the single Trp was found to be quenched on peptide binding, a signal loss that could be exploited to monitor binding in a kinetic experiment. Using NMR previously, a suggested mechanism may involve an isomerization between a predominant inactive conformation and a minority active conformation that is able to bind the peptide. In this view, the role of the phosphorylation is to shift the equilibrium between the active and inactive forms. We propose to monitor the binding kinetics using the laser T-jump technique in both resting and phosphorylated states. We are also proposing to investigate a collection of mutants with altered equilibria between the active and inactive conformation.